Efficient methods for extracting and purifying DNA are required for analyzing the biological composition (types of microorganisms, plant and animal cells) of environmental samples. Such analyses of environmental samples are performed for forensic investigations, for public health monitoring of pathogens in food and water supplies, for biological warfare agent detection, etc. A general procedure typically involves initial extraction of the DNA from cells and microorganisms in a soil, aqueous, or aerosol sample from the environment. This initial extraction may require steps such as incubation with detergent, freeze thawing, homogenization in a bead mill, and other steps. Subsequently, the DNA is analyzed or manipulated using one or more molecular biology techniques. One of the most common techniques applied to extracted DNA is amplification of particular DNA sequences by PCR (polymerase chain reaction), which uses extracted DNA molecules as templates from which exact DNA copies are made.
Environmental samples often contain materials that coextract with DNA and interfere in downstream molecular biology applications such as PCR. Known contaminants include metal ions, salts, complex polysaccharides, and protein-degrading enzymes. Contaminants that coextract with DNA from soil are poorly characterized and in most cases are unknown. Humic acids are known contaminants in soil that interfere with DNA quantitation (see, for example, Cheryl R. Kuske et al. in “Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil,” Applied and Environmental Microbiology, vol. 64, no. 7, pp. 2463–2472, July 1998; U.S. Pat. No. 6,350,578 to Peter C. Stark et al. entitled “Method of Quantitating dsDNA,” which issued on Feb. 26, 2002) inhibit PCR amplification (see, for example, Yu-Li Tsai et al. in “Detection of Low Numbers of Bacterial Cells is Soils and Sediments by Polymerase Chain Reaction,” Applied and Environmental Microbiology, vol. 58, no. 2, pp. 754–757, February 2002; Yu-Li Tsai et al. in “Rapid Method for Separation of Bacterial DNA from Humic Substances in Sediments for Polymerase Chain Reaction,” Applied and Environmental Microbiology, vol. 58, No. 7, pp. 2292–2295, July 1992; and Carol A. Kreader in “Relief of Amplification Inhibition in PCR With Bovine Serum Albumin or T4 Gene 32 Protein,” Applied and Environmental Microbiology, vol. 62, no. 3, pp. 1102–1106, March 1996) and interfere with other molecular biology applications (see, for example, Barbara J. MacGregor et al. in “Distribution and Abundance of Gram-Positive Bacteria in the Environment: Development of a Group-Specific Probe,” Journal of Microbiological Methods, vol. 44, pp. 193–203, 2001) even when present in very small concentrations.
A simple method for removing, from DNA extracts, contaminants that interfere with DNA quantitation and inhibit downstream applications such as PCR amplification is highly desirable.
Therefore, an object of the present invention is to provide a method for removing contaminants from soil samples, aqueous environmental samples, and environmental aerosol samples to obtain a purified extract for downstream applications such as PCR.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.